Liposomes have been proposed as carriers for a variety of therapeutic agents. Drug delivery systems utilizing liposomes offer the potential of improved delivery properties, including enhanced blood circulation time, reduced cytotoxicity, sustained drug release, and targeting to selected tissues.
In utilizing liposomes for drug delivery, it is generally desirable to load the liposomes to high encapsulated drug concentration. Rate of leakage of the drug from the liposomes should also be low, to preserve the advantages of drug delivery in liposome-entrapped form.
A variety of drug-loading methods are available for preparing liposomes with entrapped drug. In the case of many lipophilic drugs, efficient drug entrapment can be achieved by preparing a mixture of vesicle-forming lipids and the drug, e.g., in a dried film, and hydrating the mixture to form liposomes with drug entrapped predominantly in the lipid bilayer phase of the vesicles. Assuming the partition coefficient of the drug favors the lipid phase, high loading efficiency and stable drug retention can be achieved.
The same type of passive loading may also be employed for preparing liposomes with encapsulated hydrophilic compounds. In this case, the drug is usually dissolved in the aqueous medium used to hydrate a lipid film of vesicle-forming lipids. Depending on the hydration conditions, and the nature of the drug, encapsulation efficiencies of between about 5-20% are typically obtained, with the remainder of the drug being in the bulk aqueous phase. An additional processing step for removing non-encapsulated drug is usually required.
A more efficient method for encapsulated hydrophilic drugs, involving reverse evaporation from an organic solvent, has also been reported (Szoka, et al., 1980). In this approach, a mixture of hydrophilic drug and vesicle-forming lipids are emulsified in a water-in-oil emulsion, followed by solvent removal to form an unstable lipid-monolayer gel. When the gel is agitated, typically in the presence of added aqueous phase, the gel collapses to form oligolamellar liposomes with high (up to 50%) encapsulation of the drug.
In the case of ionizable hydrophilic or amphipathic drugs, even greater drug-loading efficiency can be achieved by loading the drug into liposomes against a transmembrane pH gradient (Nichols, et al., 1976; Cramer, et al., 1977). Typically the drug contains an ionizable amine group, and is loaded by adding it to a suspension of liposomes prepared to have a lower inside/higher outside pH gradient. Although high drug loading can be achieved by this approach (e.g., U.S. Pat. No. 5,077,056), the drug tends to leak out over time as the liposome transmembrane proton gradient decays.
The latter problem has been addressed, for drugs having an ionizable amine group, by loading the drug across an ammonium ion gradient (Haran, et al., 1993). Ammonium ions within the liposomes are in equilibrium with ammonia, which is freely permeable through the liposome membrane, and protons, which therefore accumulate as ammonia is lost from the liposomes, leading to a lower inside/higher outside pH gradient. After establishing the gradient, excess ammonium ions within the liposomes provide a reservoir of protons, to maintain the liposome pH gradient over time. This approach, however, is limited to drugs which are positively charged in their ionized state.
It would be desirable, therefore, to provide a liposome composition and method for stably loading, to high drug concentration, an ionizable drug which is negatively charged in its ionized state.